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For the most part metazoan genomes are highly methylated and harbor only small regions with low or absent methylation. In contrast, partially methylated domains (PMDs), recently discovered in a variety of cell lines and tissues, do not fit this paradigm as they show partial methylation for large portions (20%–40%) of the genome. While in PMDs methylation levels are reduced on average, we found that at single CpG resolution, they show extensive variability along the genome outside of CpG islands and DNase I hypersensitive sites (DHS). Methylation levels range from 0% to 100% in a roughly uniform fashion with only little similarity between neighboring CpGs. A comparison of various PMD-containing methylomes showed that these seemingly disordered states of methylation are strongly conserved across cell types for virtually every PMD. Comparative sequence analysis suggests that DNA sequence is a major determinant of these methylation states. This is further substantiated by a purely sequence based model which can predict 31% (R2) of the variation in methylation. The model revealed CpG density as the main driving feature promoting methylation, opposite to what has been shown for CpG islands, followed by various dinucleotides immediately flanking the CpG and a minor contribution from sequence preferences reflecting nucleosome positioning. Taken together we provide a reinterpretation for the nucleotide-specific methylation levels observed in PMDs, demonstrate their conservation across tissues and suggest that they are mainly determined by specific DNA sequence features. [ABSTRACT FROM AUTHOR]

Genomic epidemiology holds promise for malaria control and elimination efforts, for example by informing on Plasmodium falciparum genetic diversity and prevalence of mutations conferring anti-malarial drug resistance. Limited sequencing infrastructure in many malaria-endemic areas prevents the rapid generation of genomic data. To address these issues, we developed and validated assays for P. falciparum nanopore sequencing in endemic sites using a mobile laboratory, targeting key antimalarial drug resistance markers and microhaplotypes. Using two multiplexed PCR reactions, we amplified six highly polymorphic microhaplotypes and ten drug resistance markers. We developed a bioinformatics workflow that allows genotyping of polyclonal malaria infections, including minority clones. We validated the panels on mock dried blood spot (DBS) and rapid diagnostic test (RDT) samples and archived DBS, demonstrating even, high read coverage across amplicons (range: 580x to 3,212x median coverage), high haplotype calling accuracy, and the ability to explore within-sample diversity of polyclonal infections. We field-tested the feasibility of rapid genotyping in Zanzibar in close collaboration with the local malaria elimination program using DBS and routinely collected RDTs as sample inputs. Our assay identified haplotypes known to confer resistance to known antimalarials in the dhfr, dhps and mdr1 genes, but no evidence of artemisinin partial resistance. Most infections (60%) were polyclonal, with high microhaplotype diversity (median HE = 0.94). In conclusion, our assays generated actionable data within a few days, and we identified current challenges for implementing nanopore sequencing in endemic countries to accelerate malaria control and elimination., Competing Interests: The authors declare that no competing interests exist., (Copyright: © 2024 Holzschuh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)